This is a Phase II SBIR proposal to increase the throughput of our DNA sequencing instrument to enable whole genome sequencing with high efficiency and accuracy for a cost of under $1000 per genome. In Phase I, we developed a working sequencing assay, a microfluidic platform and camera system, and analysis methodology that enable genome alignment and variant calling. We have demonstrated sequencing using both synthetic DNA and genomic DNA amplicons with read lengths up over 600bp and accuracy higher than 99.9% per base. Using internal funds, we are upgrading the Phase I device to become a field ready beta test device for placement into several sites. This will be a single channel device that is capable of inline selection of 250 sequencing targets (average of 200BP long), sequence at 40X coverage, and variant calling in less than 2.5 hours of total run time. The Specific Aim of Phase II is to increase the throughput of this beta test single channel instrument to enable whole genome sequencing with high efficiency and accuracy for a cost of under $1000 per genome. To accomplish this Specific Aim, we will carry out seven Tasks: Task 1: Develop a full-genome sequencing assay protocol for the preparation of a genomic DNA library for sequencing. Task 2: Develop a sensor configuration that can scale to 100s of channels. Task 3: Develop a microfluidic device that can scale to 100s of channels Task 4: Develop a method to read droplet sizes of ~10um Task 5: Develop a sorting assay that sorts amplified DNA from empty drops Task 6: Extend current informatics pipeline for resequencing a whole genome Task 7: Extend current barcode clustering algorithms. At the conclusion of Phase II, we will have a breadboard sequencing system that will be able to demonstrate that sequencing a whole genome in about six hours, including data analysis, genome alignment and variant calling, is fully achievable. The system will support reagent volumes that will meet the per run costs target <$1000. Following Phase II, we expect to use internal funds to have a market ready product within one year. PUBLIC HEALTH RELEVANCE: In Phase II of this project, we will scale the process we have completed in our Phase I to build a machine that will be able to sequence a human genome for less than $1000. This is a project to develop a user-friendly, desktop instrument that can sequence a person's entire genetic make-up in approximately 6 hours; the entire process will cost less than $1000, including analysis. By understanding a person's genetic make-up, researchers and physicians can use the information to make personalized decisions, thereby changing the healthcare landscape and improve healthcare costs and outcomes. This instrument will greatly enhance medical treatments for tens of millions of people around the world and greatly improve our ability to understand how the body functions, which could lead to major breakthroughs in medicine.